Flexible free-standing ultrathin or thin protein membrane, its fabrication method and application

ABSTRACT

A method for fabricating a flexible free-standing ultrathin (nano) or thin protein membrane that includes (1) keeping a dilute metal (Cd, Cu or Zn) nitrate or chloride solution under neutral or weak basic pH to spontaneously form metal (Cd, Cu or Zn) hydroxide nanostrands; (2) mixing the metal (Cd, Cu or Zn) hydroxide nanostrands and protein solution to obtain composite nanofibers made of protein and the metal (Cd, Cu or Zn) hydroxide nanostrands; (3) filtering the obtained dispersion of composite nanofibers on a filter; (4) cross-linking the proteins contained in the composite nanofibers by a bifunctional cross-linker; and (5) removing the metal (Cd, Cu or Zn) hydroxide nanostrands.

This invention relates to a flexible free-standing ultrathin (nano) orthin protein membrane which enables a rapid and simple separation (orcondensation) of relatively small (M.W. ca. 1,000) molecules as well aslarge molecules. This invention also relates to a fabrication method ofthe above protein membrane and relates applications of the above proteinmembrane.

BACKGROUND OF THE INVENTION

Today, membranes are widely and practically applied to produce potablewater from sea, to clean industrial effluents and recover valuableconstituents, to concentrate, purify or fractionate macromolecularmixtures in the food and drug industries, and to separate gases andvapors. They are also key components in energy conversation systems, andin artificial organs and drug delivery devices. Their widespread use inseparations has, however, been limited by the difficulty of preparingmembranes with the desirable combination of high selectivity, whichyields high product purity and low operating costs, and highpermeability, which reduces membrane area and capital cost, as well asthe high membrane flux. Thus high membrane flux is the key performancecriterion that determines the cost of a membrane system. Unfortunately,as the selectivity of conventional polymer membrane materials increases,permeability invariably decreases and vice versa; and as decreasing thethickness to increase the flux, the stability dramatically decreased.Attempts to overcome the first fundamental limitation have explored theaddition of micron-sized porous zeolite particles to organic polymers inthe hope of combining the mechanical elasticity and processability ofpolymers with the strong size selectivity characteristic of spatiallywell-defined zeolite pores (Lai, Z. P. et al, 2003). Commercializationof this approach, however, has been hampered by poor polymer/zeoliteadhesion, inadequate particle dispersion and low membrane flux.

The developing of new nanostructured materials with specificconfigurations and morphology is offering powerful tools for thepreparation of membranes with highly controllable selectivity andpermeability for gas separation (Lai, Z. P. et al, 2003; De Vos, R. M.et al, 1998; Merekel, T. C. et al, 2002; Shiflett, M. B. et al, 1999).Up to date, nanocomposite membranes are almost keep the thickness morethan hundred nanometers and with support layer, which significantlylimit the membrane flux, separation efficiency and macroscaleapplication, especially, for liquid separation system (Holt, J. K. etal, 2006; Jirage, K. B. et al, 1997). Even several ultrathin (severaltens nanometers thick) free-standing films were reported (Yang, H. etal, 1996; Mamedov, A. A. et al, 2002; others), and used for sensors andactuators, but without any report about their separation performancebecause of the lack of the functional designation and workability,except that the first example for using ultrathin nanomembranes forsize-based macromolecular separation was carried out by Striemer' andcoworkers by using 15 nm thick free-standing silicon membranes preparedby using precision deposition of silicon and etching techniques andthermal annealing process at high temperature (above 700° C.)(Striemer.C. C. et al, 2007).

In our laboratory, we developed a general method to synthesizemacroscale ultrathin free-standing mesoporous films with fibrousnanocomposite of negatively charged dye molecules (see non-patent ref.1), DNA (see non-patent ref. 2), and positively charged metal hydroxidenanostrands (see non-patent ref. 3, non-patent ref. 4) by a simplefiltration and peeling off techniques. Unfortunately, these fibrousnanocomposite films were fragile and easily destroyed due to the weakchemical stability of metal hydroxide nanostrands. Therefore, conjugatedpolymers (polyaniline, polypyrrole) was coated on nanostrands and formedmesoporous thin films for size selective separation of proteins inphysiological conditions (Peng, X. S. et al, 2007). However, such filmstill can not be sustained in the solution with pH lower than 4.

[Non-Patent Ref. 1]

-   Luo, Y.-H., Huang, J., Ichinose, I. “Bundle-like assemblies of    cadmium hydroxide nanostrands and anionic dyes” J. Am. Chem. Soc.    127, 8296-8297 (2005).    [Non-Patent Ref. 2]-   Ichinose, I., Huang, J., Lou, Y.-H. “Electrostatic trapping of    double-strand DNA by using cadmium hydroxide nanostrands” Nano Lett.    5, 97-100 (2005).    [Non-Patent Ref. 3]-   Ichinose, I., Kurashima, K., Kunitake. T. “Spontaneous formation of    cadmium hydroxide nanostrands in water” J. Am. Chem. Soc. 126,    7162-7163 (2004).    [Non-Patent Ref. 4]-   Luo, Y.-H. et al. “Formation of positively charged copper hydroxide    nanostrands and their structural characterization” Chem. Mater. 18,    1795-1782 (2006)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

In order to solve the above problems of the fibrous nanocomposite films,we have further studied the coating of positively charged metalhydroxide nanostrands with proteins. And fortunately we have succeededin developing a robust and flexible free-standing ultrathin (nano) orthin pure protein membrane by covalently cross-linking the proteins inthe fibrous composite films by glutaraldehyde (GA) and removing away theinorganic nanostrands.

Measures for Overcoming the Problems

Namely, this invention provides a flexible free-standing ultrathin (nanoor nanometer scale) or thin protein membrane in which proteins arecross-linked (bridged) by bifunctional cross-linkers. Here, “membrane”is synonymous with “film”, and “free-standing” is synonymous with“self-supporting” in this specification.

This invention also provides a fabrication method for a protein membranewhich is a free-standing thin membrane, whose method comprises thefollowing steps: wherein “thin membrane” means “ultrathin (nano) or thinmembrane”.

(1) Step of formation of metal hydroxide nanostrands: a dilute metal(Cd, Cu or Zn) nitrate or chloride solution is kept under neutral orweak basic pH to form spontaneously metal (Cd, Cu or Zn) hydroxidenanostrands.

(2) Step of obtaining composite nanofibers made of protein and the saidmetal hydroxide nanostrands: the above metal (Cd, Cu or Zn) hydroxidenanostrands and protein solution are mixed to obtain compositenanofibers made of protein and the said metal hydroxide nanostrands;(3) Step of filtration: the obtained dispersion of composite nanofibersis filtered on a filter.(4) Step of cross-linkage: proteins contained in the compositenanofibers are cross-linked (bridged) by bifunctional cross-linkers; and(5) Step of removal of metal hydroxide nanostrands: metal (Cd, Cu or Zn)hydroxide nanostrands are removed from them.

This invention also provides some applications of the above proteinmembrane which is a free-standing thin membrane. As one of suchapplications, this invention provides a free-standing thin film composedof two layers, one of said layers being a protein membrane as definedabove, the other of said layers being a thin molecular membrane formedby depositing predetermined molecules on said protein membrane andcross-linking them by means of a bifunctional cross-linkers.

Effect of the Invention

The invented flexible free-standing ultrathin (nano) or thin proteinmembrane is novel. In some case (using apoferritin as protein), theobtained protein membranes showed the films with homogenous thickness of25 nm, and diameter of 7.5 cm, with the ratio of diameter to thicknessup to 3,000,000 (Such high ratio was not reported before).

The invented flexible free-standing ultrathin (nano) or thin proteinmembrane can be applied for size selective separation of moleculeshaving small molecular weight (ca. 1000 or less than 1000). They can bealso applied for pH controlling separation of molecules with highefficiency, and pH trigging, reversible adsorption and desorption of dyemolecules with very high capacitance of molar ratio.

According to the invented fabrication method, we can easily fabricatethe above flexible free-standing ultrathin (nano) or thin proteinmembrane.

The invented thin a free-standing film composed of two layers, one ofwhich is the above free-standing ultrathin (nano) or thin proteinmembrane, the other one is a thin molecular membrane are novelmultilayer films, and therefore they may be used differently from theinvented flexible free-standing ultrathin (nano) or thin proteinmembrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Scheme of a typical fabrication process for the ultrathin (nano)or thin free-standing protein membrane.

FIG. 2 Top view TEM image of the ferritin/cadmium hydroxide nanostrandsnanofibrous film after cross-linkage.

FIG. 3 Top view TEM image of the ferritin membrane after removal ofcadmium hydroxide nanostrands from the ferritin/cadmium hydroxidenanostrands nanofibrous film shown in FIG. 2.

FIG. 4 Copy of a photograph of a free-standing ferritin membrane with adiameter of 7.5 cm.

FIG. 5 Cross-sectional SEM image of ferritin/cadmium hydroxidenanostrands film (thickness: 40 nm) before removal of cadmium hydroxidenanostrands.

FIG. 6 Cross-sectional SEM image of ferritin membrane (thickness: 40 nm)after removal of cadmium hydroxide nanostrands.

FIG. 7 EDX spectra recorded from the cross-linked film before and afterremoval of cadmium hydroxide nanostrands.

FIG. 8 FTIR spectra of the film of (i) before cross-linkage and (ii)after cross-linkage followed by removal of cadmium hydroxidenanostrands.

FIG. 9 Typical loading-unloading curves of ferritin and apoferritinfilms with and without cadmium nanostrands.

FIG. 10 Typical UV-visible spectra and copies of photographs showingconcentration performance for PC/Cu.

FIG. 11 Typical UV-visible spectra and copies of photographs showingabsorption of Evans Blue by using apoferritin membrane (300 nm thick,3.2 cm diameter).

FIG. 12 Typical UV-visible spectra and copies of photographs showingdesorption of Evans Blue by using apoferritin membrane (300 nm thick,3.2 cm diameter).

FIG. 13 Typical photoluminescence spectra showing that the fluorescentdye molecules adsorbed by ferritin membrane. The inset photograph (copy)was taken under irradiated with 375 nm light. The molecular structurewas shown in the down inset.

FIG. 14 Typical SEM images of a PAMAM membrane (thickness: 4.6micrometer) formed on a apoferritin membrane (thickness: 1.9 micrometer)surface. (b) shows magnified image of parts surrounded by dotted line in(a).

MEANING OF SYMBOL

(In FIG. 9)

-   -   1: Apoferritin with nanostrands    -   2: Apoferritin without nanostrands    -   3: Ferritin with nanostrands    -   4: Ferritin without nanostrands

BEST MODE FOR CARRYING OUT THE INVENTION

Firstly, we explain a fabrication method for the free-standing ultrathin(nano) or thin protein membrane in detail. The invented fabricationmethod comprises the following steps as mentioned above.

(1) Step of formation of metal (Cd, Cu or Zn) hydroxide nanostrands;

(2) Step of obtaining composite nanofibers made of protein and the saidmetal hydroxide nanostrands;

(3) Step of filtration;

(4) Step of cross-linkage; and

(5) Step of removal of metal hydroxide nanostrands.

Though we are not needed to process in the above order, but the aboveorder, namely (1)→(2)→(3)→(4)→(5), is most preferable.

Furthermore, we can add an additional step of peeling-off of compositenanofibers made of protein and the metal hydroxide nanostrands after (4)step of cross-linkage.

We can widely use various kinds of protein in this invention. Later weshow examples using ferritin, apoferritin, cytochrome c, myoglobin andglucose oxidase (needless to say, the other protein can also be used).Mixed proteins can also be used as well as single protein, but singleprotein is preferable because we expect a good uniformity of membrane.

A scheme of a typical fabrication process for the free-standingultrathin (nano) or thin protein membrane was shown in FIG. 1.

In the initial step (i.e. formation of metal hydroxide nanostrands; notillustrated here), polymer-like positively charged metal hydroxidenanostrands are prepared as we described elsewhere (non-patent ref. 3,4). Briefly, pH of a dilute Cd, Cu or Zn nitrate (otherwise, Cd or Znchloride) solution is raised up to neutral or weak basic (pH=6.0-8.5) byadding a dilute alkali solution, and kept it at room temperature forseveral minutes to one day resulting in spontaneous formation of metalhydroxide nanostrands, whose diameter is ca. 2-3 nm and whose lengthreaches several tens of micrometers.

The obtained metal hydroxide nanostrands are mixed with negativelycharged protein solution under stirring for defined hours, resulting indispersion of composite nanofibers made of protein and the metalhydroxide nanostrands. The obtained dispersion is filtered on a filtersuch as polycarbonate (PC) membrane filter with 200 nm pores (porosityabout 10%), forming composite nanofibrous films. Then, the films aretreated with a solution of bifunctional cross-linkers (such as 10 wt %glutaraldehyde aqueous solution) for sufficient hours to completecross-linkage reaction.

In case of using glutaraldehyde, the reaction is as followed.Protein-NH₂+O═CHC₃H₆HC═O→Protein-N═CHC₃H₆HC═N-protein+2H₂O

We can also use other bifunctional cross-linkers, for example, variousimidoesters, N-hydroxysuccinimide-esters or carbodiimides which are wellknown to be bifunctional cross-linkers of proteins, in substitution forglutaraldehyde.

In FIG. 1, an example that these cross-linked nanofibrous compositefilms are peeled off is shown. In order to peel off, we can immerse theabove filter with films in alcohol (e.g. ethanol) resulting in formingcross-linked free-standing films.

Subsequently the above cross-linked free-standing films can be immersedin aqueous mineral acid such as HCl solution to remove the metalhydroxide nanostrands. Then the excess metal ions and HCl can be washedaway using purified water. Thus we can obtain the pure (namely, notcontaining metal hydroxide) free-standing protein membranes floating inwater. We can store these membranes in alcohol for further applicationand characterization.

The thickness of the protein membrane can be easily controlled from 10nm to 10 μm by adjusting the volume of the filtering fibrous compositesolution (see Example 3, Table 1).

In order to execute a rapid and simple separation (or condensation) oftarget materials, the thickness of the protein membrane is preferably 15nm˜1000 nm, and more preferably 20 nm˜1000 nm. When a definedconcentration of the fibrous composite are filtered, the thickness ofthe protein membrane and the time of the filtering process linearlydepend on the filtering volume.

The diameter of the protein membrane is not limited, because thediameter of the film is basically determined by the inner diameter ofthe filtering funnel.

The above free-standing ultrathin or thin protein membrane has variousapplications. As described above, one application is a preparation ofthin free-standing films composed of two layers, one of which is theabove protein membrane, the other one is a thin molecular membrane whichis formed by stacking defined molecules on the said protein membrane andcross-linking with bifunctional cross-linkers.

Here, as the defined molecules we can use various defined molecules suchas synthetic macromolecules whose molecular weight is large enough notto go through the channels of the above protein membrane. We can use,for example, dendrimers having terminal NH₂ groups as the said syntheticmacromolecules. Then, the dendrimers are preferably polyamidoamine whosemolecular weight is over ca. 2,000 (large enough not to go through thechannels of the above protein membrane).

EXAMPLES

Materials used in the following examples are followed. CdCl₂.2.5H₂O,Cu(NO₃)₂.3H₂O, 2-aminoethanol, Direct Yellow 50, Evans Blue,8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt, K₃[Fe(CN)₆],hydrogen chloride (5 M solution), glutaraldehyde (50 wt % aqueoussolution) were purchased from Kanto Chemical.Tetrakis(1-methylpyridinium-4-yl) porphine p-Toluenesulfonate,8-octanoyloxypyrene-1,3,6-trisulfonic acid trisodium salt, copperphthalocyanine tetrasulfonic acid tetrasodium salt, glucose oxidase,cytochrome c, myoglobin, horse spleen ferritin (76 mg/ml solution), andapoferritin (38 mg/ml solution) were purchased from Sigma Aldrich.Deionized water (18.2 MΩ) was produced by a Millipore Direct-Q System,and used throughout the experiments. Polycarbonate (PC) membrane andfilters (Nuclepore, Whatman) of 2.5 cm, 4.7 cm, and 9.0 cm in diameterwere used for the preparation of free-standing films. Alumina membranes(Anodise, pore size 0.2 μm, diameter 2.5 cm, thickness 60 μm) were alsopurchased from Whatman.

Instruments and methods used are followed. The films were characterizedby using a scanning electron microscope (SEM, Hitachi S-4800), atransmission electron microscope (TEM, JEOL 1010), and a high-resolutiontransmission electron microscopy (HR-TEM, JEM 2100F) equipped with anenergy-dispersive X-ray analysis system. The specimens for TEM andHR-TEM observation were prepared by transferring the free-standing filmon a carbon-coated TEM grid. SEM observation was conducted after coating2-nm thick platinum layer by using a Hitachi e-1030 ion sputter at thepressure of 10 Pa and the current density of 10 mA. UV-vis absorptionspectra were obtained by using a SHIMAZU UV-3150 spectrophotometer. Thephotoluminescence spectra were obtained by a JASCO FP-6500spectrofluorometer. The magnetic properties were measured using acommercial magnetometer with superconducting interference devise(MPMS-XL, Quantum Design). Mechanical properties were measured by usingTriboIndenter (Hysitron Inc.), employing a diamond Berkovitch indenterwith silicon substrate. The molecular dimensions estimated by a Chem3Dultra 10.0 (Cambridge Scientific Computing).

Example 1 Preparation and Characterization of the Invented ProteinMembrane

(a) Preparation

In the initial step, polymer-like positively charged cadmium hydroxidenanostrands were prepared as we described elsewhere (non-patent ref.2-4). Briefly, cadmium hydroxide nanostrands were prepared by quicklymixing a dilute NaOH or aminoethanol solution (2 mM, 20 mL) into 20 mLof 4 mM aqueous cadmium nitrate and stirring for a few minutes.

Protein (ferritin, apoferritin, cytochrome c, myoglobin, or glucoseoxidase) was added into the above dispersion of cadmium hydroxidenanostrands and mixed under stirring for 30 mins. In the case offerritin and apoferritin, the mixture was made of 1 ml, 3.8 mg/mlprotein solution and 20 ml cadmium hydroxide nanostrand solution. In thecase of cytochrome c, myoglobin, and glucose oxidase, 1 ml, 6.4 mg/mlprotein solution mixed with 20 ml cadmium hydroxide nanostrand solution.

A certain volume of the mixture was filtered on polycarbonate membranes(diameters of membrane/funnel used for filtration: 3.2 cm) by suctionfiltering process under gauge pressure (ΔP) of 90 KPa. Then, the filmswere immersed into 10 wt % glutaraldehyde aqueous solution and crosslinked for 1 hour at room temperature. These cross-linked nanofibrouscomposite films were peeled off by immersing PC membrane with films inethanol. The resulting free-standing films were immersed in 10 mM HClsolution for 3 hrs to remove the inorganic nanostrands and then theexcess cadmium ions and HCl were washed away using milli-Q water. Thuswe obtained five kinds of pure free-standing protein (ferritin,apoferritin, cytochrome c, myoglobin, and glucose oxidase) membranesfloating in water respectively.

(b) Characterization

FIG. 2 is TEM image of the ferritin/cadmium hydroxide nanostrandsnanofibrous film after cross-linkage and this clearly shows that fibrousstructures and the proteins almost assembled along the nanostrands. Inthis image, the ferritin proteins appear as black dots with diameterabout 8 nm due to the iron compound cores of ferritin. The cadmiumhydroxide nanostrands appear as about 2 nm fiber structures.

FIG. 3 is TEM image of the pure ferritin membrane after removal of metalhydroxide nanostrands from the ferritin/cadmium hydroxide nanostrandsnanofibrous film shown in FIG. 2, and this shows that the fibrousstructures were disappeared, which means that the nanostrands werecompletely removed.

FIG. 4 shows a copy of the photograph of a representative free-standingferritin membrane with diameters of 7.5 cm. The diameter of the membraneis equal to the size of the funnel using for filtration.

The morphology and composition of the film before and after removal ofcadmium hydroxide nanostrands were further examined in detail by usingSEM and EDX. Free-standing films were transferred onto anodic aluminamembranes with pore size 200 nm.

FIG. 5 shows (a) cross-sectional and (b) top view SEM image offerritin/cadmium hydroxide nanostrands film (thickness: 40 nm) beforeremoval of cadmium hydroxide nanostrands, and FIG. 6 shows (a)cross-sectional and (b) top view SEM image of ferritin membrane(thickness: 40 nm) after removal of cadmium hydroxide nanostrands.

Comparing FIG. 5 a and FIG. 6 a, the thickness of the films didn't showbig difference, namely, there was almost no decrease of thickness afterremoval of nanostrands. This means that there is no collapse due to theremoval of nanostrands. But the surface of the film after removal ofnanostrands becomes smoother than that of before removal of nanostrands(compare FIG. 5 b and FIG. 6 b).

FIG. 7 shows EDX spectra recorded from the cross-linked film before andafter removal of cadmium hydroxide nanostrands. These EDX spectraconfirmed that the cadmium element was completely removed away from thefilm which is consistent with the above TEM investigations. The FTIRspectra of native ferritin (no cross-linkage) and the obtained pureferritin membrane were shown in FIG. 8. The characteristic peaks arealmost the same, just the intensity of the peaks of ferritin membraneare stronger than that of native ferritin. The same position of peaks isbecause no any new functional group was introduced into the membraneduring cross-linking process. The increasing of the intensity of thepeaks at about 1660 cm⁻¹ is originated from C═N covalent bond formationduring the cross-linking process (Rozkiewicz, D. I. et al, Chem. Eur. J.12, 6290-6297, 2006). These results indicate that the protein is notdenatured.

Additionally, even though cadmium hydroxide nanostrands do not affectthe protein membrane due to removal of them, the other more safenanostrands, such as copper hydroxide nanostrands and zinc hydroxidenanostrands with similar properties to that of cadmium hydroxidenanostrands, were also used successfully to prepare free-standingprotein membranes (data were not shown here).

Example 2 Controlled Synthesis of Different Thickness and DiameterProtein Membrane

(a) Preparation

We prepared ultrathin free-standing pure protein membranes similarly toExample 1 except that the time of the filtering process and/or thefiltering volume were varied and that diameters of membrane/funnel usedfor filtration were 1.7 cm, 3.2 cm and 7.5 cm. The results were shown inTable 1.

TABLE 1 Process V_(mixture) ^(a) time Diameter Thickness Error ProteinNo. (ml) (min) (cm) (nm) (nm) Ferritin 1 0.25 0.5 1.7 40 ±2 2 0.375 0.751.7 60 ±2 3 0.65 1.2 1.7 105 ±5 4 1.25 3 1.7 200 ±5 5 3.75 8 1.7 600 ±106 10 20 1.7 1,550 ±20 7 25 60 1.7 4,000 ±50 8 1.7 1.2 3.2 60 ±2 9 9 103.2 300 ±10 10 9 1 7.5 60 ±2 Apo- 11 10 11 3.2 300 ±10 ferritin 12 3.50.5 7.5 25 ±2 13 7 1 7.5 45 ±2 14 20 3 7.5 140 ±5 Myoglobin 15 0.25 0.51.7 50 ±2 Cyt. c 16 0.3 0.5 1.7 50 ±2 GOx 17 0.3 0.5 1.7 50 ±2 Here,V_(mixture) ^(a) is the volume of the filtered mixture, which was madeof 2 ml, 3.8 mg/ml of ferritin or apoferritin protein(or 6.4 mg/ml ofother protein) solution and 40 ml cadmium hydroxide nanostrandssolution. The thickness of protein membrane was measured from thecross-section SEM images.(b) Characterization

The films were characterized by SEM images and TEM images (not shownhere) as well as by naked eye's observation (or photographs). From theseresults we can see that the thickness of the protein membrane and thetime of the filtering process linearly depend on the filtering volume.In these examples, thin films were synthesized in three diameters, 1.7cm, 3.2 cm and 7.5 cm. And we also found for ferritin, the thinnestthickness is 40 nm for 1.7 cm, but which is 60 nm for 3.2 and 7.5 cm,respectively. The thickest one can be reach up to 4000 nm for 1.7 cmwith one hour filtering time. Namely, in case of ferritin the thicknessof ferritin films can be controlled in the range from 40 nm to 4,000 nm.

Comparing the example 60 nm, 200 nm and 600 nm, and 4000 nm thick filmsbefore removal of cadmium nanostrands, the thicker the film, the deeperthe color was. The thicknesses of the films of corresponding proteinfilms after removal of cadmium hydroxide nanostrands were almost notchanged. This indicates that the films were not clasped after removal ofnanostrands, resulting more porous films.

In case of another protein, apoferritin, the obtained apoferritinultrathin protein membranes showed the films with homogenous thicknessof 25 nm, even in the case of 7.5 cm diameter (No. 12). The ratio ofdiameter to thickness is up to 3,000,000. TEM image (not shown here)indicates the film is very flexible in nanometer scale. The extremelymacroscale flexibility was confirmed by the aspiration of this 7.5 cmdiameters apoferritin free-standing membranes into a pipette tip withdiameter of 0.8 mm. The protein membranes surprisingly could reversiblypass through a holes 8,790 times smaller than its own area. This is dueto its flexible and extreme thinness.

The mechanical properties of ferritin and apoferritin films withthickness 1550 nm before and after removal of cadmium hydroxidenanostrands were measured by nanoindentation using TriboIndenter(Hysitron Inc.), employing a diamond Berkovitch indenter, respectively.The typical load-unload curves for them were shown in FIG. 9. For eachsample, three points were measured and giving the average data. Thehardness, H, and Young's modules were shown in Table 2.

TABLE 2 Mechanical properties of ferritin and apoferritin films E.Young's module (GPa)^(a) Hardness (MPa) Sample n1^(b) n2 n3 Avg.^(c) n1n2 n3 Avg. Ferritin 5.97 6.20 6.25 6.14 378 386 391 385 (before)^(d)Ferritin 4.29 4.41 4.63 4.44 327 326 313 322 (after)^(e) Apoferritin4.49 5.06 4.75 4.77 316 349 315 327 (before) Apoferritin 4.43 4.69 4.644.58 312 310 308 310 (after) ^(a)the value of Poisson ration is 0.5 andused for calculating the Young's module. ^(b)n1 means the measurementpoint number. n1, n2 and n3, means three points are measured. ^(c)Avgmeans average value of that measured from three points. ^(d)(before)means before removing cadmium hydroxide nanostrands. ^(e)(after) meansafter removing cadmium hydroxide nanostrands.

It can be concluded that hardness and the Young's modules of filmsbefore removal of nanostrands are larger than that of the films afterremoval of nanostrands. At the same time, the hardness and Young'smodules of ferritin films are larger than that of apoferritin. Thedifference among the films before and after removal of nanostrands showsthat the enhanced mechanical properties of the protein films areoriginated from interaction between the inorganic components andprotein. At the same time, the iron compound in ferritin makes theferritin film with larger value of hardness and Young's modules thanthat of apoferritin film. These values of the hardness and the Young'smodules of ferritin and apoferritin films are ten times larger than thatreported of glyoxal cross-linked native protein films, such as gelatin,soy, casein and sodium caseinate (Vaz, C. M. et al, J. Mater. Sci.:Mater. in Medicine 14, 789-796, 2003), and 4 times as that ofglutaraldehyde cross-linked soy protein films (Chabba, S. et al, J.Mater. Sci. 40, 6263-6273, 2005).

We additionally did an experiment that a 60 nm thick film was sealed ona plastic tube hole with diameter inner 5 mm and outer diameter 7 mm,then connected with another plastic tube with inner diameter 7 mm andouter diameter 10 mm, and then ethanol solution of directly yellow wascarefully filled into the large tube and hold it vertically. It wasfound that such film can support a column of ethanol of 21.5 cm (it wascalculated that about 180,000 times heavier than its own weight wassupported without apparent permeation of ethanol).

When the nanostrands were removed from the ferritin or apoferritin film,the film becomes more flexible as seen by naked eye observation of filmswith 1550 nm thick (photographs not shown). This phenomenon is inagreement with the mechanical properties before and after removenanostrands. These protein films after removal of nanostrands are verystable under both acid and basic conditions, as well as for organicsolvent solution, such as acetone, benzene and chloroform, which aredesirable for application.

Example 3 Application of the Invented Protein Membrane to Separation,Condensation, Absorption or Desorption

The separation performance of these free-standing ultrathin pure proteinmembranes were investigated by studying the permeation of molecules withdifferent size, charge states, and pH. The filtration process wascarried out under 90 kPa pressure. The volume of the molecules is keptas 20 ml. The flux rate is equal to the permeated volume divided by thevalid area and the processing time. The permeation performance wasmonitored by the UV-Vis absorption spectra recorded on the solutionbefore and after filtration, and also the upper solution. The resultswere summarized in Table 3.

TABLE 3 The separation performance of different molecules at differentpH through a 60 nm thin free-standing ferritin membranes Molecules C L ×W^(a) Molecule- Ferritin- Permeation Flux rate (mw) (μM) pH (Å²)Charging charging (%) Lm⁻²h⁻¹ ANTS 40 6.27 − − 99.86 7639.4 (650.58)1.53 8.2 × 7.0 − + 98.75 7370.3 13.3 − − 99.67 9167.3 [Fe(CN)₆]³⁻ 10006.25 8.7 × 8.7 − − 99.63 7545.6 (212) EB 10 6.41 28.5 × 7.8  − − 27.987065.6 (960.81) 13.1 − − 99.54 8653.4 PC/Cu 2 1.44 18.6 × 13.4 − + 0.096374.3 (984.25) 13.01 − − 0.14 8356.4 6.75 − − 0.1 6945.2 TMPyP 1.5 6.3218.2 × 13.2 + − 0.15 6354.7 (1,363.6) Cyc. c 2 mg/ml 6.39 2.5 × 2.5 ×3.7 + − 0.06 6276.9 (13,000) 11.02 − − 0.04 6512.4 DY 10 1.66 29.3 ×8.1  − + 0.55 5372.4 (956.82) 2.71 − + 0.81 5382.6 3.19 − + 0.95 5364.23.83 − + 25.13 5400.2 4.31 − + 25.38 6122.3 6.19 − − 33.51 6178.5 7.09 −− 99.32 7258.9 9.53 − − 99.53 7268.4 12.11 − − 99.12 7350.3 12.96 − −99.96 8767.5 DY/(PC/Cu) 10/2 6.35 29.3 × 8.1/  − − 43.51/ 6985.1 0.0513.12 1.61 × 1.61 − − 99.45/ 8546.7 0.04

High performance size-based selective molecular separation was achievedby using a 60 nm thin, ferritin film with diameter of 1.7 cm. Bigmolecules, such as PC/Cu (MW:984.25), TMPyP dye (MW:1,363.6) and Cyt. c(MW:13,000) were completely separated from their aqueous solutions. ANTSmolecules (MW:650.58) and [Fe(CN)₆]³⁺ ions (MW:212) completely wentthrough such film. DY (direct yellow 50) (MW:956.82), and EB (Evansblue) (MW:960.81) molecules partially permeated through it at neutralpH. However, by changing the pH of DY or EB solutions, these dyemolecules completely went through the protein membrane at pH higher than7 and were forbidden to pass at pH lower than 3.19. These pH dependentperformances are due to the surface charge property of the protein. Inthe case for ferritin and apoferritin, the isoelectric points of themare the same at about pH 4.4. So, when the pH of dye solution is lowerthan 4.4, the membrane is positively charged, and adsorbs the negativelycharged dye molecules through electrostatic interaction, and makes thechannels smaller. If the dye molecules are smaller enough, such as ANTSmolecules, 8.23×7.0×3.17 Å³ and [Fe(CN)₆]³; 8.7×8.7×8.7 Å³, they stillcan go through the smaller channels. However, if the molecule size isbigger than these smaller channels, it can not pass through the film. Ofcause, if the dye molecules are bigger than the channels, at any pH, itcan not go through the film. At pH 13.03, the permeation of the mixturesolution of PC/Cu and DY molecules shows that DY thoroughly permeatesthrough the film, but PC/Cu were almost can't pass through it. However,all the flux rates of water are more than 5,000 Lm⁻² h⁻¹ under pressureof 90 kPa. Due to no permeation of the bigger molecules, they can beconcentrated from their diluted solution.

For example, PC/Cu molecules solution was examined (FIG. 10). PC/Cumolecules could be efficiently concentrated from 10 μM solution to 28.89μM within 2 min by using a 60 nm thick ferritin membrane on aluminamembrane by suck filtration under 90 kPa. UV-vis spectra were used tomonitor the efficiency of the concentration performance. Increasing theprocess time, the upper solution become more and more blue, but there isno detectable PC/Cu in the filtration solution. The flux rate is 6671.9L·m⁻² h⁻¹.

The reversible charged properties of the proteins, when the pH is higheror lower than its isoelectric point, they can be used as an absorber forthe charged molecules or particles in solution with different pH, andthen release them into the other solution by changing the pH. For thisstudy, the reversible absorption and desorption of dye molecules (Evansblue and DY) were investigated. 300 nm thick and 3.2 cm diameterapoferritin membrane was used for adsorption and desorption of Evansblue molecules in solution by controlling the pH of the solution. Thedye molecules in 10 ml, 10 μM, Evans blue solution at pH 1.50 can becompletely captured by the protein membrane within 1 day. And thesecaptured molecules can be further released in basic solution such as pH13.09 water. The capturing and releasing process were monitored byUv-vis spectroscopy.

FIG. 11 a shows typical UV-visible spectra recorded during the capturingprocess with increasing time. FIG. 11 b, copies of photographs oforiginal and after one day, clearly show that the dye molecules werecompletely removed from the solution by the membrane and this wasconsistent with the Uv-vis result. The releasing performance was shownin FIG. 12 a and FIG. 12 b. After 2.5 hrs, 83.5% of Evans blue moleculeswere released from the membrane into the solution. The longer the time,the more amount dye molecules was released into the solution. Themaximum rate of the first released circle is 94.5% similar to that of DYmolecules (data not shown). But, after the first capture and releasingcircle, the release efficiency is 99.4% for the later circles. Thecapture and release process is due to the pH triggering the chargeproperties of the apoferritin membrane. When the pH of the solution islower than that isoelectric of apoferritin pH 4.4, the membrane will bepositively charged, contrary; if the pH higher than 4.4, the proteinmembrane will be negatively charged. Therefore, in the pH of thesolution lower than 4.4, negatively charged molecules will be trappedinto the membrane through electrostatic interaction, otherwise, thenegatively charged dye molecules will be push away from the membrane dueto the repulsion force. The blue color (FIG. 11) and purple color (FIG.12) of the Evans blue solution with pH 1.50 and 13.09, respectively isdue to the dye molecules itself.

Another example for the adsorption performance was shown in FIG. 13.Fluorescent dye, 8-octanoyloxypyrene-1,3,6-trisulfonic acid trisodiumsalt (OPTAT), 10 μM, 10 ml solution with pH 1.39, were adsorbed by 300nm thick, 3.2 cm diameter, ferritin membrane. The photoluminescence (PL)spectra show that after 7 hours, all the molecules were trapped into theferritin membrane, and the film showed stronger emission than that ofthe original solution. The inset photo was obtained by exciting themembrane at 375 nm.

Example 4

Another application of the invented protein membrane to preparation ofthin free-standing films composed of two layers, the one layer is theprotein membrane, the other layer is a thin molecular membrane formed onit.

(a) Preparation of protein (apoferritin) films: the process ofpreparation of protein (apoferritin) films was the same as described inthe Example 1.

(b) Preparation of thin free-standing films composed of two layers, theone layer is the protein (apoferritin) membrane, the other layer is athin molecular membrane formed on the protein (apoferritin) membranesurface

A 1.9 micrometer thin apoferritin membrane mounted on a polycarbonatemembrane with pore size of 200 nm was used in a suction filtrationsystem as filter. 2 ml, 0.2 wt % dendrimer, PAMAM (Polyamidoamine)molecule (general 4, molecular weight: 14215, diameter: 4.5 nm, surface—NH₂ groups: 64, purchased from Sigma-Aldrich) methanol solution wasfiltered on the above system under 90 kPa pressure. After filtering awaymethanol, PAMAM molecule filter cake was cross-linked by 1 ml, 5 wt %glutaraldehyde for 1.5 hrs. Finally, the film was washed by methanol andwater four times. FIG. 14 shows typical SEM images of a 4.6 micrometerthick PAMAM film formed on a 1.9 micrometer thick apoferritin filmsurface.

The thickness of the PAMAM film can be controlled by the filteringvolume of PAMAM solution.

Additionally other molecular films also can be prepared by using thesame filtering and cross-linking process based on protein membranes.This method provides a simple way to build functional thin molecularfilms for various applications, such as separation.

What we claim is:
 1. A fabrication method for a protein membrane, whichis a flexible free-standing thin membrane, the method comprising: (1)keeping a dilute metal (Cd, Cu or Zn) nitrate or chloride solution underneutral or weak basic pH to form spontaneously metal (Cd, Cu or Zn)hydroxide nanostrands whose diameters are 2-3 nm; (2) mixing the metal(Cd, Cu or Zn) hydroxide nanostrands and protein solution therebyobtaining a dispersion of composite nanofibers made of protein and themetal (Cd, Cu or Zn) hydroxide nanostrands; (3) filtering the dispersionof the composite nanofibers on a filter; (4) cross-linking the proteincontained in the composite nanofibers by a bifunctional cross-linkerthereby obtaining a cross-linked nanofibrous composite film; and (5)removing the metal (Cd, Cu or Zn) hydroxide nanostrands with thediameters of 2-3 nm from the cross-linked nanofibrous composite filmthereby obtaining a flexible free-standing thin protein membranecontaining channels.
 2. The fabrication method according to claim 1,wherein said bifunctional cross-linker is glutaraldehyde.
 3. Thefabrication method according to claim 1, wherein the protein solutioncontains a protein selected from the group consisting of ferritin,apoferritin, cytochrome c, myoglobin, and glucose oxidase.
 4. Thefabrication method according to claim 1, further comprising peeling thecross-linked nanofibrous composite film before the removing of the metal(Cd, Cu or Zn) hydroxide nanostands.
 5. The fabrication method accordingto claim 1, wherein the protein membrane has the channels whosediameters are 2-3 nm.
 6. The fabrication method according to claim 1,wherein the filter is a polycarbonate membrane.
 7. The fabricationmethod according to claim 1, wherein the protein membrane has apressure-resistance property.
 8. The fabrication method according toclaim 1, wherein a length of the metal (Cd, Cu or Zn) hydroxidenanostrands is several tens of micrometers.